Determining how abused drugs affect neurological function requires us to advance our basic understanding of the structure and operation of the synaptic cleft. Traditional tools in neuroscience, which measure averaged properties of bulk samples, have contributed an immense amount of information toward this goal. To understand fully the synapse, however, we must develop new tools of unprecedented sensitivity, tools that are capable of analyzing single synaptic components and observing conformational changes during vesicle fusion at the synapse. The objective of this proposal is to develop such tools and techniques. We plan to use capillary electrophoresis, optical trapping, controlled vesicle fusion, fluorescence resonance energy transfer, biosensor detection, laser induced fluorescence, and single-molecule detection to investigate: (a) the contents of individual synaptic vesicles, and (b) the structure and function of individual proteins involved in the process of vesicle fusion with the cell membrane in the synaptic cleft. In addition to yielding valuable information about the synaptic vesicle cycle and exocytosis, the purpose of this research program is to establish a valuable set of techniques that can be used by the neuroscience community in their investigations of synaptic transmission.